Microtubule-associated protein

ABSTRACT

The invention provides a human microtubule-associated protein (HMAP) and polynucleotides which identify and encode HMAP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HMAP.

[0001] This application is a DIVISIONAL of pending prior applicationU.S. Ser. No. 09/013,118, filed on Jan. 26, 1998, entitledMICROTUBULE-ASSOCIATED PROTEIN.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a human microtubule-associated protein and to the use of thesesequences in the diagnosis, treatment, and prevention of diseasesassociated with immune disorders and cancer.

BACKGROUND OF THE INVENTION

[0003] Microtubules (MTs) are involved in many cellular functions suchas mitosis, morphogenesis, motility, and intracellular organelletransport. For example, disruption of MTs by various MT-destabilizingagents results in major changes in cytoplasmic organization, includingcollapse of intermediate filaments and redistribution of the Golgiapparatus and endoplasmic reticulum. MTs also function in themaintenance of cell shape, movement of eukaryotic cilia and flagella,formation of the mitotic spindle, and regulation of organelledistribution and vesicle movements. Regulation of the dynamic propertiesof MTs is believed to play a role in many of these functions.

[0004] A group of proteins that bind MTs are defined asmicrotubule-associated proteins (MAPs). MAPS are involved in modulatingMT dynamics and function. MAPs can be categorized into two major classesaccording to their primary function; motor proteins, which include thekinesin superfamily and the dynein family, and non-motor proteins, whichare further divided into the high molecular weight MAPs, includingMAP1A, MAP1B, MAP2, and MAP4 and the low molecular weight tau proteins.(See, e.g., Ding M. et al. (1996) J. Biol. Chem. 271:12555-12561.) Amongthe identified functions of MAPs in neuronal cells, kinesin andcytoplasmic dynein are responsible for anterograde and retrograde axonaltransport, respectively. Tau protein and MAP2 appear to be required forinitial neurite growth. It is well known that the function of MAPs canbe regulated through phosphorylation. For example, phosphorylation ofMAPs can alter their affinity for MTs in vivo and affect their abilityto stabilize MTs.

[0005] Hyperphosphorylated microtubule-associated protein tau is themajor proteinaceous component of paired helical and straight filamentswhich constitute a defining neuropathological characteristic ofAlzheimer's disease and a number of other neurodegenerative disorders.Full length tau protein can assemble into Alzheimer-like filaments uponincubation with heparin, a repeating disaccharide chain. Heparin alsopromotes phosphorylation of tau protein by a number of kinases, preventstau protein from binding to taxol-stabilized microtubules, and producesrapid disassembly of microtubules assembled from tau protein andtubulin. Moreover, cAMP-dependent protein kinase andCa²⁺/calmodulin-dependent protein kinase II phosphorylate tau proteinsat specific sites in vitro, some of which are also phosphorylated inPHF-tau. Hyperphosphorylation of tau protein results in its inability tobind to microtubules and is believed to precede paired helical filamentassembly. Phosphorylation-independent interaction between recombinanttau protein and sulfated glycosaminoglycans leads to the formation ofAlzheimer-like filaments under physiological conditions in vitro. (Seee.g., Hasegawa, M. Et al. (1997) J. Biol. Chem. 272:33118-33124.)

[0006] The discovery of a new human microtubule-associated protein andthe polynucleotides encoding it satisfies a need in the art by providingnew compositions which are useful in the diagnosis, treatment, andprevention of diseases associated with cell proliferation.

SUMMARY OF THE INVENTION

[0007] The invention features a substantially purified polypeptide,human microtubule-associated protein (HMAP), comprising a sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0008] The invention further provides a substantially purified variantof HMAP having at least 90% amino acid identity to the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide encoding the polypeptide comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionalso includes an isolated and purified polynucleotide variant having atleast 90% polynucleotide identity to the polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

[0009] Additionally, the invention provides a composition comprising apolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1. The invention further provides anisolated and purified polynucleotide which hybridizes under stringentconditions to the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide encoding the polypeptide comprising the sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

[0010] The invention also provides an isolated and purifiedpolynucleotide comprising a sequence of SEQ ID NO:2 or a fragment of SEQID NO:2, and an isolated and purified polynucleotide variant having atleast 90% polynucleotide identity to the polynucleotide comprising thesequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention alsoprovides an isolated and purified polynucleotide which is complementaryto the polynucleotide comprising the sequence of SEQ ID NO:2 or afragment of SEQ ID NO:2.

[0011] The invention further provides an expression vector containing atleast a fragment of the polynucleotide encoding the polypeptidecomprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Inanother aspect, the expression vector is contained within a host cell.

[0012] The invention also provides a method for producing a polypeptidecomprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideencoding HMAP under conditions suitable for the expression of thepolypeptide; and (b) recovering the polypeptide from the host cellculture.

[0013] The invention also provides a pharmaceutical compositioncomprising a substantially purified HMAP having the sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

[0014] The invention further includes a purified antibody which binds toa polypeptide comprising the sequence of SEQ ID NO:1 or a fragment ofSEQ ID NO:1, as well as a purified agonist and a purified antagonist ofthe polypeptide.

[0015] The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist to HMAP.

[0016] The invention also provides a method for treating or preventingan immune disorders, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist to HMAP.

[0017] The invention also provides a method for detecting apolynucleotide encoding HMAP in a biological sample containing nucleicacids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one ofthe nucleic acids of the biological sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding HMAP in the biological sample. Inone aspect, the nucleic acids of the biological sample are amplified bythe polymerase chain reaction prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the amino acid sequence (SEQID NO:1) and nucleic acid sequence (SEQ ID NO:2) of HMAP. The alignmentwas produced using MACDNASIS PRO software (Hitachi Software EngineeringCo. Ltd., San Bruno, Calif.).

[0019]FIGS. 2A and 2B show the amino acid sequence alignments among HMAP(1312429; SEQ ID NO:1) and microtubule-associated protein fromSaccharomyces cerevisiae (GI 1928989; SEQ ID NO:3), produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.).

[0020]FIGS. 3A and 3B show the hydrophobicity plots for HMAP, SEQ IDNO:1 and microtubule-associated protein from S. cerevisiae (SEQ IDNO:3), respectively; the positive X axis reflects amino acid position,and the negative Y axis, hydrophobicity (MACDNASIS PRO software).

DESCRIPTION OF THE INVENTION

[0021] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0022] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, vectors, and methodologies which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

[0024] Definitions

[0025] “HMAP,” as used herein, refers to the amino acid sequences ofsubstantially purified HMAP obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0026] The term “agonist,” as used herein, refers to a molecule which,when bound to HMAP, increases or prolongs the duration of the effect ofHMAP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of HMAP.

[0027] An “allele” or an “allelic sequence,” as these terms are usedherein, is an alternative form of the gene encoding HMAP. Alleles mayresult from at least one mutation in the nucleic acid sequence and mayresult in altered mRNAs or in polypeptides whose structure or functionmay or may not be altered. Any given natural or recombinant gene mayhave none, one, or many allelic forms. Common mutational changes whichgive rise to alleles are generally ascribed to natural deletions,additions, or substitutions of nucleotides. Each of these types ofchanges may occur alone, or in combination with the others, one or moretimes in a given sequence.

[0028] “Altered” nucleic acid sequences encoding HMAP, as describedherein, include those sequences with deletions, insertions, orsubstitutions of different nucleotides, resulting in a polynucleotidethe same HMAP or a polypeptide with at least one functionalcharacteristic of HMAP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding HMAP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding HMAP. The encoded protein may also be “altered,” and maycontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentHMAP. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of HMAP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

[0029] The terms “amino acid” or “amino acid sequence,” as used herein,refer to an oligopeptide, peptide, polypeptide, or protein sequence, ora fragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments”, “immunogenic fragments”, or“antigenic fragments” refer to fragments of HMAP which are preferablyabout 5 to about 15 amino acids in length and which retain somebiological activity or immunological activity of HMAP. Where “amino acidsequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

[0030] “Amplification,” as used herein, relates to the production ofadditional copies of a nucleic acid sequence. Amplification is generallycarried out using polymerase chain reaction (PCR) technologies wellknown in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler(1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., pp. 1-5.)

[0031] The term “antagonist,” as it is used herein, refers to a moleculewhich, when bound to HMAP, decreases the amount or the duration of theeffect of the biological or immunological activity of HMAP. Antagonistsmay include proteins, nucleic acids, carbohydrates, antibodies, or anyother molecules which decrease the effect of HMAP.

[0032] As used herein, the term “antibody” refers to intact molecules aswell as to fragments thereof, such as Fa, F(ab′)₂, and Fv fragments,which are capable of binding the epitopic determinant. Antibodies thatbind HMAP polypeptides can be prepared using intact polypeptides orusing fragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0033] The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

[0034] The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term “antisense strand” is used in referenceto a nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

[0035] As used herein, the term “biologically active,” refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic HMAP, or ofany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0036] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

[0037] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding HMAP orfragments of HMAP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0038] The phrase “consensus sequence,” as used herein, refers to anucleic acid sequence which has been resequenced to resolve uncalledbases, extended using the XL-PCR kit (Perkin Elmer, Norwalk, Conn.) inthe 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from the overlapping sequences of more than one Incyte Cloneusing a computer program for fragment assembly, such as the GELVIEWfragment assembly system (GCG, Madison, Wis.). Some sequences have beenboth extended and assembled to produce the consensus sequence.

[0039] As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HMAP, bynorthern analysis is indicative of the presence of nucleic acidsencoding HMAP in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding HMAP.

[0040] A “deletion,” as the term is used herein, refers to a change inthe amino acid or nucleotide sequence that results in the absence of oneor more amino acid residues or nucleotides.

[0041] The term “derivative,” as used herein, refers to the chemicalmodification of HMAP, of a polynucleotide sequence encoding HMAP, or ofa polynucleotide sequence complementary to a polynucleotide sequenceencoding HMAP. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

[0042] The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

[0043] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MegAlign program (LASERGENE softwarepackage, DNASTAR, Inc., Madison Wis.). The MegAlign program can createalignments between two or more sequences according to different methods,e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The Clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be calculated by theClustal Method, or by other methods known in the art, such as the JotunHein Method. (See, e.g., Hein, J. (1990) Methods in Enzymology183:626-645.) Identity between sequences can also be determined by othermethods known in the art, e.g., by varying hybridization conditions.

[0044] “Human artificial chromosomes” (HACs), as described herein, arelinear microchromosomes which may contain DNA sequences of about 6 kb to10 Mb in size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

[0045] The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

[0046] “Hybridization,” as the term is used herein, refers to anyprocess by which a strand of nucleic acid binds with a complementarystrand through base pairing.

[0047] As used herein, the term “hybridization complex” as used herein,refers to a complex formed between two nucleic acid sequences by virtueof the formation of hydrogen bonds between complementary bases. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or formed between one nucleic acid sequence present insolution and another nucleic acid sequence immobilized on a solidsupport (e.g., paper, membranes, filters, chips, pins or glass slides,or any other appropriate substrate to which cells or their nucleic acidshave been fixed).

[0048] The words “insertion” or “addition,” as used herein, refer tochanges in an amino acid or nucleotide sequence resulting in theaddition of one or more amino acid residues or nucleotides,respectively, to the sequence found in the naturally occurring molecule.

[0049] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0050] The term “microarray,” as used herein, refers to an array ofdistinct polynucleotides or oligonucleotides arrayed on a substrate,such as paper, nylon or any other type of membrane, filter, chip, glassslide, or any other suitable solid support.

[0051] The term “modulate,” as it appears herein, refers to a change inthe activity of HMAP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HMAP.

[0052] The phrases “nucleic acid” or “nucleic acid sequence,” as usedherein, refer to an oligonucleotide, nucleotide, polynucleotide, or anyfragment thereof, to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

[0053] The terms “operably associated” or “operably linked,” as usedherein, refer to functionally related nucleic acid sequences. A promoteris operably associated or operably linked with a coding sequence if thepromoter controls the transcription of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in reading frame, certain genetic elements, e.g.,repressor genes, are not contiguously linked to the encoded polypeptidebut still bind to operator sequences that control expression of thepolypeptide.

[0054] The term “oligonucleotide,” as used herein, refers to a nucleicacid sequence of at least about 6 nucleotides to 60 nucleotides,preferably about 15 to 30 nucleotides, and most preferably about 20 to25 nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimers,”“primers,” “oligomers,” and “probes,” as these terms are commonlydefined in the art.

[0055] “Peptide nucleic acid” (PNA), as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least about 5 nucleotides in length linked to a peptide backboneof amino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

[0056] The term “sample,” as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acids encoding HAP,or fragments thereof, or HMAP itself may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

[0057] As used herein, the terms “specific binding” or “specificallybinding” refer to that interaction between a protein or peptide and anagonist, an antibody, or an antagonist. The interaction is dependentupon the presence of a particular structure of the protein recognized bythe binding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

[0058] As used herein, the term “stringent conditions” refers toconditions which permit hybridization between polynucleotide sequencesand the claimed polynucleotide sequences. Suitably stringent conditionscan be defined by, for example, the concentrations of salt or formamidein the prehybridization and hybridization solutions, or by thehybridization temperature, and are well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0059] For example, hybridization under high stringency conditions couldoccur in about 50% formamide at about 37° C. to 42° C. Hybridizationcould occur under reduced stringency conditions in about 35% to 25%formamide at about 30° C. to 35° C. In particular, hybridization couldoccur under high stringency conditions at 42° C. in 50% formamide,5×SSPE, 0.3% SDS, and 200 μg/ml sheared and denatured salmon sperm DNA.Hybridization could occur under reduced stringency conditions asdescribed above, but in 35% formamide at a reduced temperature of 35° C.The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Variations on the above ranges and conditions are well known in the art.

[0060] The term “substantially purified,” as used herein, refers tonucleic acid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

[0061] A “substitution,” as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0062] “Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

[0063] A “variant” of HMAP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0064] The Invention

[0065] The invention is based on the discovery of a new humanmicrotubule-associated protein (HMAP), the polynucleotides encodingHMAP, and the use of these compositions for the diagnosis, treatment, orprevention of immune disorders and cancer.

[0066] Nucleic acids encoding the HMAP of the present invention werefirst identified in Incyte Clone 1312429 from the bladder tumor cDNAlibrary (BLADTUT02) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 1312429 (BLADTUT02), 2368182 and 2368151 (ADRENOT07), 3519656(LUNGNON03), 3730457 (SMCCNON03), 1260511 (SYNORAT05), 216842(STOMNOT01), and 1214331 (BRSTTUT01).

[0067] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, 1C, 1D, 1E, and 1F. HMAP is 423 amino acids in length and has onepotential cAMP- and cGMP-dependent protein kinase phosphorylation siteat residue S₁₅₃; three potential casein kinase II phosphorylation sitesat residues S₇₆, T₂₂₁, and S₂₆₆; nine potential protein kinase Cphosphorylation sites at residues S₁₀₆, T₁₂₂, T₂₃₁, T₂₇₉, S₂₈₉, T₃₁₇,S₃₄₇, S₃₇₄, and S₄₁₁; one potential tyrosine kinase phosphorylation siteat residue R₇; and two potential beta-transduction family trp-asprepeats signature sites beginning at residues I₁₂₇, and L₃₅₈ in whichW₂₇₆D and W₃₇₀D are the trp-asp repeat motifs. As shown in FIGS. 2A and2B, HMAP has chemical and structural homology withmicrotubule-associated protein from S. cerevisiae (GI 1928989; SEQ IDNO:3) and the W₃₇₀D repeat domain. In particular, HMAP andmicrotubule-associated protein from S. cerevisiae share 32% identity. Asillustrated by FIGS. 3A and 3B, HMAP and microtubule-associated proteinfrom S. cerevisiae have rather similar hydrophobicity plots. A usefulfragment of the nucleic acid sequence (SEQ ID NO:2) is from aboutC₃₁₆-C₃₄₃ and T₄₅₁-C₄₇₂. Northern analysis shows the expression of thissequence in various libraries, at least 63% of which are immortalized orcancerous and at least 21% of which involve the immune response.

[0068] The invention also encompasses HMAP variants. A preferred HMAPvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the HMAP amino acid sequence, and which contains at leastone functional or structural characteristic of HMAP.

[0069] The invention also encompasses polynucleotides which encode HMAP.In a particular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an HMAP.

[0070] The invention also encompasses a variant of a polynucleotidesequence encoding HMAP. In particular, such a variant polynucleotidesequence will have at least about 80%, more preferably at least about90%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding HMAP. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of HMAP.

[0071] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding HMAP, some bearing minimal homology tothe polynucleotide sequences of any known and naturally occurring gene,may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringHMAP, and all such variations are to be considered as being specificallydisclosed.

[0072] Although nucleotide sequences which encode HMAP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HMAP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HMAP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HMAP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0073] The invention also encompasses production of DNA sequences whichencode HMAP and HMAP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding HMAP or any fragment thereof.

[0074] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:2, or a fragment of SEQID NO:2, under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; and Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.)

[0075] Methods for DNA sequencing are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nev.), PTC200 thermal cycler (MJ Research, Watertown,Mass.) and the ABI CATALYST and 373 and 377 DNA sequencers (PerkinElmer).

[0076] The nucleic acid sequences encoding HMAP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCRMethods Applic. 2:318-322.) In particular, genomic DNA is firstamplified in the presence of a primer complementary to a linker sequencewithin the vector and a primer specific to the region predicted toencode the gene. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0077] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia, T. etal. (1988) Nucleic Acids Res. 16:8186.) The primers may be designedusing commercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0078] Another method which may be used is capture PCR, which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060.) Additionally, one mayuse PCR, nested primers, and PROMOTERFINDER libraries (Clontech, PaloAlto, Calif.) to walk genomic DNA. This process avoids the need toscreen libraries and is useful in finding intron/exon junctions.

[0079] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0080] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and a charge coupled device camera for detection of theemitted wavelengths. Output/light intensity may be converted toelectrical signal using appropriate software (e.g., GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer), and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0081] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode HMAP may be used in recombinant DNAmolecules to direct expression of HMAP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHMAP.

[0082] As will be understood by those of skill in the art, it may beadvantageous to produce HMAP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0083] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterHMAP-encoding sequences for a variety of reasons including, but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0084] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HMAP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HMAP activity, it may be useful toencode a chimeric HMAP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HMAP encoding sequence and theheterologous protein sequence, so that HMAP may be cleaved and purifiedaway from the heterologous moiety.

[0085] In another embodiment, sequences encoding HMAP may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res.Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp.Ser. 225-232.) Alternatively, the protein itself may be produced usingchemical methods to synthesize the amino acid sequence of HMAP, or afragment thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995)Science 269:202-204.) Automated synthesis may be achieved using the ABI431A peptide synthesizer (Perkin Elmer).

[0086] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or by sequencing. (See, e.g., Creighton, T. (1983) Proteins,Structures and Molecular Properties, W H Freeman and Co., New York,N.Y.) Additionally, the amino acid sequence of HMAP, or any partthereof, may be altered during direct synthesis and/or combined withsequences from other proteins, or any part thereof, to produce a variantpolypeptide.

[0087] In order to express a biologically active HMAP, the nucleotidesequences encoding HMAP or derivatives thereof may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0088] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding HMAPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

[0089] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding HMAP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0090] The “control elements” or “regulatory sequences” are thosenon-translated regions, e.g., enhancers, promoters, and 5′ and 3′untranslated regions, of the vector and polynucleotide sequencesencoding HMAP which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters, e.g., hybrid lacZpromoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) orPSPORT1 plasmid (GIBCO/BRL), may be used. The baculovirus polyhedrinpromoter may be used in insect cells. Promoters or enhancers derivedfrom the genomes of plant cells (e.g., heat shock, RUBISCO, and storageprotein genes) or from plant viruses (e.g., viral promoters or leadersequences) may be cloned into the vector. In mammalian cell systems,promoters from mammalian genes or from mammalian viruses are preferable.If it is necessary to generate a cell line that contains multiple copiesof the sequence encoding HMAP, vectors based on SV40 or EBV may be usedwith an appropriate selectable marker.

[0091] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HMAP. For example, whenlarge quantities of HMAP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HMAP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced, and pIN vectors. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors(Pharmacia Biotech, Uppsala, Sweden) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0092] In the yeast Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters, such as alpha factor,alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel, supra; andGrant et al. (1987) Methods Enzymol. 153:516-544.)

[0093] In cases where plant expression vectors are used, the expressionof sequences encoding HMAP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews. (See, e.g., Hobbs,S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

[0094] An insect system may also be used to express HMAP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding HMAPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of sequences encoding HMAP will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses may then be used to infect, forexample, S. frugiperda cells or Trichoplusia larvae in which HMAP may beexpressed. (See, e.g., Engelhard, E. K. et al. (1994) Proc. Nat. Acad.Sci. 91:3224-3227.)

[0095] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding HMAP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing HMAP in infected host cells. (See, e.g.,Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) Inaddition, transcription enhancers, such as the Rous sarcoma virus (RSV)enhancer, may be used to increase expression in mammalian host cells.

[0096] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

[0097] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding HMAP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding HMAP and its initiation codon and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.Cell Differ. 20:125-162.)

[0098] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

[0099] For long term, high yield production of recombinant proteins,stable expression is preferred. For example, cell lines capable ofstably expressing HMAP can be transformed using expression vectors whichmay contain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0100] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase genes and adeninephosphoribosyltransferase genes, which can be employed in tk⁻ or apr⁻cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; npt confers resistance to the aminoglycosides neomycin andG-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additionalselectable genes have been described, e.g., trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine. (See, e.g., Hartman, S. C. andR. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Recently,the use of visible markers has gained popularity with such markers asanthocyanins, β glucuronidase and its substrate GUS, luciferase and itssubstrate luciferin. Green fluorescent proteins (GFP) (Clontech, PaloAlto, Calif.) are also used (See, e.g., Chalfie, M. et al. (1994)Science 263:802-805.) These markers can be used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system. (See, e.g.,Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131.)

[0101] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding HMAP is inserted within a marker gene sequence, transformedcells containing sequences encoding HMAP can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding HMAP under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0102] Alternatively, host cells which contain the nucleic acid sequenceencoding HMAP and express HMAP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

[0103] The presence of polynucleotide sequences encoding HMAP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding HMAP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding HMAP todetect transformants containing DNA or RNA encoding HMAP.

[0104] A variety of protocols for detecting and measuring the expressionof HMAP, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HMAP is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).

[0105] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HMAPinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding HMAP, or any fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byPharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S.Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules orlabels which may be used for ease of detection include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

[0106] Host cells transformed with nucleotide sequences encoding HMAPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode HMAP may be designed to contain signal sequences which directsecretion of HMAP through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding HMAP tonucleotide sequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the HMAP encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing HMAP and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography). (See, e.g., Porath, J. et al. (1992) Prot.Exp. Purif. 3: 263-281.) The enterokinase cleavage site provides a meansfor purifying HMAP from the fusion protein. (See, e.g., Kroll, D. J. etal. (1993) DNA Cell Biol. 12:441-453.)

[0107] Fragments of HMAP may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, T. E. (1984) Protein: Structures andMolecular Properties, pp. 55-60, W. H. Freeman and Co., New York, N.Y.)Protein synthesis may be performed by manual techniques or byautomation. Automated synthesis may be achieved, for example, using anAP 431A peptide synthesizer (Perkin Elmer). Various fragments of HMAPmay be synthesized separately and then combined to produce the fulllength molecule.

[0108] Therapeutics

[0109] Chemical and structural homology exists between HMAP andmicrotubule-associated protein from S. cerevisiae (GI 1928989). Inaddition, HMAP is expressed in tissues associated with immune disordersand cancer. Therefore, HMAP appears to play a role in immune responseand cancer.

[0110] Therefore, in one embodiment, an antagonist of HMAP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a cancer. Such cancers may include, but are not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma; and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds HMAP may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissues which express HMAP.

[0111] In another embodiment, a vector expressing the complement of thepolynucleotide encoding HMAP may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

[0112] Therefore, in another embodiment, an antagonist of HMAP may beadministered to a subject to prevent or treat an immune disorder. Immunedisorders may include, but are not limited to AIDS, Addison's disease,adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis, andextracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma. In one aspect, anantibody which specifically binds HMAP may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissues which express HMAP.

[0113] In another embodiment, a vector expressing the complement of thepolynucleotide encoding HMAP may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedescribed above.

[0114] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0115] An antagonist of HMAP may be produced using methods which aregenerally known in the art. In particular, purified HMAP may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind HMAP. Antibodies to HMAP may alsobe generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0116] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HMAP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0117] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to HMAP have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofHMAP amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0118] Monoclonal antibodies to HMAP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0119] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HMAP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

[0120] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

[0121] Antibody fragments which contain specific binding sites for HMAPmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab)2 fragments. Alternatively, Fab expression librariesmay be constructed to allow rapid and easy identification of monoclonalFab fragments with the desired specificity. (See, e.g., Huse, W. D. etal. (1989) Science 246:1275-1281.)

[0122] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between HMAP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HMAP epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

[0123] In another embodiment of the invention, the polynucleotidesencoding HMAP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HMAP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding HMAP. Thus, complementary molecules or fragments may be used tomodulate HMAP activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments can be designed from variouslocations along the coding or control regions of sequences encodingHMAP.

[0124] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors which will express nucleic acidsequences complementary to the polynucleotides of the gene encodingHMAP. (See, e.g., Sambrook, supra; and Ausubel, supra.)

[0125] Genes encoding HMAP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding HMAP. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0126] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding HMAP. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0127] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHMAP.

[0128] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0129] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding HMAP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0130] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0131] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15:462-466.)

[0132] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0133] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of HMAP, antibodies to HMAP, and mimetics, agonists,antagonists, or inhibitors of HMAP. The compositions may be administeredalone or in combination with at least one other agent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0134] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0135] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0136] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0137] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0138] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0139] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0140] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0141] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0142] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0143] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0144] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of HMAP, such labeling wouldinclude amount, frequency, and method of administration.

[0145] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0146] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0147] A therapeutically effective dose refers to that amount of activeingredient, for example HMAP or fragments thereof, antibodies of HMAP,and agonists, antagonists or inhibitors of HMAP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ED50/LD50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0148] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0149] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0150] Diagnostics

[0151] In another embodiment, antibodies which specifically bind HMAPmay be used for the diagnosis of disorders characterized by expressionof HMAP, or in assays to monitor patients being treated with HMAP oragonists, antagonists, or inhibitors of HMAP. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for HMAP include methods whichutilize the antibody and a label to detect HMAP in human body fluids orin extracts of cells or tissues. The antibodies may be used with orwithout modification, and may be labeled by covalent or non-covalentattachment of a reporter molecule. A wide variety of reporter molecules,several of which are described above, are known in the art and may beused.

[0152] A variety of protocols for measuring HMAP, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of HMAP expression. Normal or standard valuesfor HMAP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HMAP under conditions suitable for complex formation Theamount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of HMAP expressedin subject, control, and disease samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0153] In another embodiment of the invention, the polynucleotidesencoding HMAP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof HMAP may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of HMAP, and tomonitor regulation of HMAP levels during therapeutic intervention.

[0154] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HMAP or closely related molecules may be used to identifynucleic acid sequences which encode HMAP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HMAP, alleles, orrelated sequences.

[0155] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the HMAP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the HMAP gene.

[0156] Means for producing specific hybridization probes for DNAsencoding HMAP include the cloning of polynucleotide sequences encodingHMAP or HMAP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or 35S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

[0157] Polynucleotide sequences encoding HMAP may be used for thediagnosis of a disorder associated with expression of HMAP. Examples ofsuch a disorder include, but are not limited to, a cancer such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma; and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and an immune disorder suchas AIDS, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, and extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections; and trauma. The polynucleotide sequences encoding HMAP maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered HMAP expression. Such qualitative orquantitative methods are well known in the art. The polynucleotidesequences encoding HMAP may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered HMAP expression. Suchqualitative or quantitative methods are well known in the art.

[0158] In a particular aspect, the nucleotide sequences encoding HMAPmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding HMAP may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding HMAP in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0159] In order to provide a basis for the diagnosis of a disorderassociated with expression of HMAP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding HMAP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0160] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0161] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0162] Additional diagnostic uses for oligonucleotides designed from thesequences encoding HMAP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HMAP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HMAP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0163] Methods which may also be used to quantitate the expression ofHMAP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and interpolating results from standardcurves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) Thespeed of quantitation of multiple samples may be accelerated by runningthe assay in an ELISA format where the oligomer of interest is presentedin various dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

[0164] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0165] In one embodiment, the microarray is prepared and used accordingto methods known in the art. (See, e.g., Chee et al. (1995) PCTapplication WO95/11995; Lockhart, D. J. et al. (1996) Nat. Biotech.14:1675-1680; and Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619.)

[0166] The microarray is preferably composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs. The oligonucleotidesare preferably about 6 to 60 nucleotides in length, more preferablyabout 15 to 30 nucleotides in length, and most preferably about 20 to 25nucleotides in length. It may be preferable to use oligonucleotideswhich are about 7 to 10 nucleotides in length. The microarray maycontain oligonucleotides which cover the known 5′ or 3′ sequence,sequential oligonucleotides which cover the full length sequence, orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest.Oligonucleotides can also be specific to one or more unidentified cDNAsassociated with a particular cell type or tissue type. It may beappropriate to use pairs of oligonucleotides on a microarray. The firstoligonucleotide in each pair differs from the second oligonucleotide byone nucleotide. This nucleotide is preferably located in the center ofthe sequence. The second oligonucleotide serves as a control. The numberof oligonucleotide pairs may range from about 2 to 1,000,000.

[0167] In order to produce oligonucleotides for use on a microarray, thegene of interest is examined using a computer algorithm which starts atthe 5′ end, or, more preferably, at the 3′ end of the nucleotidesequence. The algorithm identifies oligomers of defined length that areunique to the gene, have a GC content within a range suitable forhybridization, and lack secondary structure that may interfere withhybridization. In one aspect, the oligomers may be synthesized on asubstrate using a light-directed chemical process. (See, e.g., Chee etal., supra.) The substrate may be any suitable solid support, e.g.,paper, nylon, any other type of membrane, or a filter, chip, or glassslide.

[0168] In another aspect, the oligonucleotides may be synthesized on thesurface of the substrate using a chemical coupling procedure and an inkjet application apparatus. (See, e.g., Baldeschweiler et al. (1995) PCTapplication WO95/251116.) An array analogous to a dot or slot blot(HYBRIDOT apparatus, GIBCO/BRL) may be used to arrange and link cDNAfragments or oligonucleotides to the surface of a substrate using avacuum system or thermal, UV, mechanical, or chemical bondingprocedures. An array may also be produced by hand or by using availabledevices, materials, and machines, e.g. Brinkmann multichannel pipettorsor robotic instruments. The array may contain from 2 to 1,000,000 or anyother feasible number of oligonucleotides.

[0169] In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a sample. The sample may be obtainedfrom any bodily fluid, e.g., blood, urine, saliva, phlegm, gastricjuices, cultured cells, biopsies, or other tissue preparations. Toproduce probes, the polynucleotides extracted from the sample are usedto produce nucleic acid sequences complementary to the nucleic acids onthe microarray. If the microarray contains cDNAs, antisense RNAs (aRNAs)are appropriate probes. Therefore, in one aspect, mRNA isreverse-transcribed to cDNA. The cDNA, in the presence of fluorescentlabel, is used to produce fragment or oligonucleotide aRNA probes. Thefluorescently labeled probes are incubated with the microarray so thatthe probes hybridize to the microarray oligonucleotides. Nucleic acidsequences used as probes can include polynucleotides, fragments, andcomplementary or antisense sequences produced using restriction enzymes,PCR, or other methods known in the art.

[0170] Hybridization conditions can be adjusted so that hybridizationoccurs with varying degrees of complementarity. A scanner can be used todetermine the levels and patterns of fluorescence after removal of anynonhybridized probes. The degree of complementarity and the relativeabundance of each oligonucleotide sequence on the microarray can beassessed through analysis of the scanned images. A detection system maybe used to measure the absence, presence, or level of hybridization forany of the sequences. (See, e.g., Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. 94:2150-2155.)

[0171] In another embodiment of the invention, nucleic acid sequencesencoding HMAP may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev.7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)

[0172] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) MolecularBiology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding HMAP on a physical chromosomalmap and a specific disorder, or a predisposition to a specific disorder,may help define the region of DNA associated with that disorder. Thenucleotide sequences of the invention may be used to detect differencesin gene sequences among normal, carrier, and affected individuals.

[0173] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., AT to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0174] In another embodiment of the invention, HMAP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HMAPand the agent being tested may be measured.

[0175] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with HMAP, orfragments thereof, and washed. Bound HMAP is then detected by methodswell known in the art. Purified HMAP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0176] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding HMAPspecifically compete with a test compound for binding HMAP. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with HMAP.

[0177] In additional embodiments, the nucleotide sequences which encodeHMAP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0178] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0179] I. BLADTUT02 cDNA Library Construction

[0180] The BLADTUT02 cDNA library was constructed from cancerous bladdertissue from an 80-year-old Caucasian female who had undergone radicalcystectomy following diagnosis of grade 3 (of 4) invasive transitionalcell carcinoma forming a mass on the posterior wall of the bladder withextension into the trigone. The tumor was found to be deeply invasive,measuring up to 1 cm in thickness, extending to perivesical fat andextending to within 0.8 cm to the vaginal mucosal margin. Left pelviclymph node was found negative for tumor. Patient's history includeddiagnoses of malignant neoplasm of uterus, benign hypertension,atherosclerosis, and atrial fibrillation. Prior to surgery, the patienthad undergone bladder operation, total abdominal hysterectomy, removalof both ovaries, partial thyroidectomy, division of thyroid isthmus,aortocoronary bypass of three coronary arteries, and resection andreplacement of abdominal aorta. Patient's family history includeddiagnoses of acute renal failure and osteoarthritis in patient's mother,and atherosclerosis in patient's father and sibling.

[0181] The frozen tissue was homogenized and lysed using a PT-3000homogenizer polytron (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using a SW28 rotor in a L8-70M ultracentrifuge(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.The RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-freewater, and DNase treated at 37° C. RNA extraction and precipitation wereas before. The mRNA was then isolated using the OLIGOTEX mRNApurification kit (QIAGEN, Inc.; Chatsworth, Calif.) and used toconstruct the cDNA library.

[0182] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT plasmid system (Gibco/BRL). The cDNAs were fractionatedon a SEPHAROSE CL4B column (Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pINCY 1. The plasmid pINCY 1 was subsequentlytransformed into DH5α competent cells (Gibco/BRL).

[0183] II. Isolation and Sequencing of cDNA Clones

[0184] Plasmid DNA was released from the cells and purified using theR.E.A.L PREP 96 plasmid purification kit (QIAGEN, Inc.). This kitenabled the simultaneous purification of 96 samples in a 96-well blockusing multi-channel reagent dispensers. The recommended protocol wasemployed except for the following changes: 1) the bacteria were culturedin 1 ml of sterile Terrific Broth (GIBCO/BRL) with carbenicillin at 25mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0185] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:441f), using a MICROLAB 2200 liquid transfer system(Hamilton, Reno, Nev.) in combination with PTC200 thermal cyclers (M JResearch, Watertown, Mass.) and 377 or 373 DNA sequencing systems(Perkin Elmer).

[0186] III. Homology Searching of cDNA Clones and Their Deduced Proteins

[0187] After the reading frame was determined, the nucleotide sequencesof the Sequence Listing or amino acid sequences deduced from them wereused as query sequences against databases such as GenBank, SwissProt,BLOCKS, and Pima II. These databases which contain previously identifiedand annotated sequences, were searched for regions of homology(similarity) using BLAST, which stands for Basic Local Alignment SearchTool (Altschul (1993) supra, Altschul (1990) supra).

[0188] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms such as the one described in Smith et al. (1992, ProteinEngineering 5:35-51), incorporated herein by reference, could have beenused when dealing with primary sequence patterns and secondary structuregap penalties. The sequences disclosed in this application have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

[0189] The BLAST approach, as detailed in Karlin et al. (supra) andincorporated herein by reference, searched for matches between a querysequence and a database sequence. BLAST evaluated the statisticalsignificance of any matches found, and reported only those matches thatsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

[0190] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam); and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology.

[0191] The nucleotide sequences and/or amino acid sequences of theSequence Listing were used to query sequences in the GenBank, SwissProt,BLOCKS, and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

[0192] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. (See, e.g., Smith, T. etal. (1992) Protein Engineering 5:35-51.) The sequences disclosed in thisapplication have lengths of at least 49 nucleotides and have no morethan 12% uncalled bases (where N is recorded rather than A, C, G, or T).

[0193] The BLAST approach searched for matches between a query sequenceand a database sequence. BLAST evaluated the statistical significance ofany matches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

[0194] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and other mammalian sequences(mam), and deduced amino acid sequences from the same clones were thensearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

[0195] IV. Northern Analysis

[0196] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; and Ausubel, F. M. et al. supra, ch. 4 and 16.)

[0197] Analogous computer techniques applying BLAST are used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Pharmaceuticals). This analysis ismuch faster than multiple membrane-based hybridizations. In addition,the sensitivity of the computer search can be modified to determinewhether any particular match is categorized as exact or homologous.

[0198] The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}{\quad \quad}{identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0199] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Homologous molecules are usually identified by selecting thosewhich show product scores between 15 and 40, although lower scores mayidentify related molecules.

[0200] The results of northern analysis are reported as a list oflibraries in which the transcript encoding HMAP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0201] V. Extension of HMAP Encoding Polynucleotides

[0202] The nucleic acid sequence of Incyte Clone 1312429 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension of an antisense polynucleotide, and the other was synthesizedto initiate extension of a sense polynucleotide. Primers were used tofacilitate the extension of the known sequence “outward” generatingamplicons containing new unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 software (National Biosciences, Plymouth, Minn.), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

[0203] Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0204] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. PCR was performed using the PTC200 thermalcycler (M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94°C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72° C.for 8 min Step 13 4° C. (and holding)

[0205] A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using the QIAQUICK DNA purification system(QIAGEN Inc., Chatsworth, Calif.), and trimmed of overhangs using Klenowenzyme to facilitate religation and cloning.

[0206] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing 2×Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2×Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

[0207] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4for an additional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0208] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0209] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0210] VI. Labeling and use of Individual Hybridization Probes

[0211] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences) and labeled by combining 50pmol of each oligomer, 250 μCi of [γ-³²P] adenosine triphosphate(Amersham, Chicago, Ill.), and T4 polynucleotide kinase (DuPont NEN,Boston, Mass.). The labeled oligonucleotides are substantially purifiedusing a SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn,Kalamazoo, Mich.). An aliquot containing 10⁷ counts per minute of thelabeled probe is used in a typical membrane-based hybridization analysisof human genomic DNA digested with one of the following endonucleases:Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II (DuPont NEN, Boston,Mass.).

[0212] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots for several hours,hybridization patterns are compared visually.

[0213] VII. Microarrays

[0214] To produce oligonucleotides for a microarray, one of thenucleotide sequences of the present invention is examined using acomputer algorithm which starts at the 3′ end of the nucleotidesequence. For each, the algorithm identifies oligomers of defined lengththat are unique to the nucleic acid sequence, have a GC content within arange suitable for hybridization, and lack secondary structure thatwould interfere with hybridization. The algorithm identifiesapproximately 20 oligonucleotides corresponding to each nucleic acidsequence. For each sequence-specific oligonucleotide, a pair ofoligonucleotides is synthesized in which the first oligonucleotidesdiffers from the second oligonucleotide by one nucleotide in the centerof the sequence. The oligonucleotide pairs can be arranged on asubstrate, e.g. a silicon chip, using a light-directed chemical process.(See, e.g., Chee, supra.)

[0215] In the alternative, a chemical coupling procedure and an ink jetdevice can be used to synthesize oligomers on the surface of asubstrate. (See, e.g., Baldeschweiler, supra.) An array analogous to adot or slot blot may also be used to arrange and link fragments oroligonucleotides to the surface of a substrate using or thermal, UV,mechanical, or chemical bonding procedures, or a vacuum system. Atypical array may be produced by hand or using available metods andmachines and contain any appropriate number of elements. Afterhybridization, nonhybridized probes are removed and a scanner used todetermine the levels and patterns of fluorescence. The degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray may be assessed through analysis of thescanned images.

[0216] VIII. Complementary Polynucleotides

[0217] Sequences complementary to the HMAP-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HMAP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of HMAP. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HMAP-encoding transcript.

[0218] IX. Expression of HMAP

[0219] Expression of HMAP is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., β-galactosidase upstreamof the cloning site, operably associated with the cDNA of interest.(See, e.g., Sambrook, supra, pp. 404-433; and Rosenberg, M. et al.(1983) Methods Enzymol. 101: 123-138.)

[0220] Induction of an isolated, transformed bacterial strain withisopropyl beta-D-thiogalactopyranoside (IPTG) using standard methodsproduces a fusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of HMAP into bacterialgrowth media which can be used directly in the following assay foractivity.

[0221] X. Demonstration of HMAP Activity

[0222] Phosphorylation assays for HMAP (0.050 ml) are carried out at 30°C. and comprise 25 mM Tris-HCl, pH 7.4, 0.1 mM EGTA, 0.1 mM sodiumorthovanadate, 2.5 uM PKI (a specific inhibitor of cyclic AMP-dependentprotein kinase), protease inhibitors (0.5 mM phenylmethylsulfonylfluoride, 5 ug/ml aprotinin, 5 ug/ml leupeptin, and 0.5 ug/mlpepstatin), HMAP (4 uM), 10 mM magnesium acetate, 2 mM [gamma-³²P]ATP(approximately 10⁶ cpm/nmol), and 1 unit/ml activated p42 MAP kinase, 1unit/ml GSK3, or 5 units/ml recombinant reconstituted neuronalcdc-2-like kinase. Glycosaminoglycans and nucleic acids are included inthe assays at 50 ug/ml and tubulin at 20 uM. Reactions are initiated byaddition of ATP and aliquots are removed at various times ranging from10 minutes to 24 hours and used for SDS-polyacrylamide gelelectrophoresis and immunoblotting. Alternatively, incorporation of ³²Pradioactivity is measured after absorption to Whatman P-81 paper. (Seee.g., Hasegawa, M. Et al. (1997) J. Biol. Chem. 272:33118-33124.)

[0223] XI. Production of HMAP Specific Antibodies

[0224] HMAP substantially purified using PAGE electrophoresis (see,e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or otherpurification techniques, is used to immunize rabbits and to produceantibodies using standard protocols. The HMAP amino acid sequence isanalyzed using DNASTAR software (DNASTAR Inc) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Methods for selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions are well described in the art.(See, e.g., Ausubel et al. supra, ch. 11.)

[0225] Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel et al. supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity, for example, bybinding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

[0226] XII. Purification of Naturally Occurring HMAP Using SpecificAntibodies

[0227] Naturally occurring or recombinant HMAP is substantially purifiedby immunoaffinity chromatography using antibodies specific for HMAP. Animmunoaffinity column is constructed by covalently coupling anti-HMAPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0228] Media containing HMAP are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of HMAP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HMAP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HMAPis collected.

[0229] XIII. Identification of Molecules Which Interact with HMAP

[0230] HMAP, or biologically active fragments thereof, are labeled with¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HMAP, washed, and anywells with labeled HMAP complex are assayed. Data obtained usingdifferent concentrations of HMAP are used to calculate values for thenumber, affinity, and association of HMAP with the candidate molecules.

[0231] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 3 1 423 PRT Homo sapiens misc_feature Incyte ID No 1312429 1 Met AlaGln Leu Gln Thr Arg Phe Tyr Thr Asp Asn Lys Lys Tyr 1 5 10 15 Ala ValAsp Asp Val Pro Phe Ser Ile Pro Ala Ala Ser Glu Ile 20 25 30 Ala Asp LeuSer Asn Ile Ile Asn Lys Leu Leu Lys Asp Lys Asn 35 40 45 Glu Phe His LysHis Val Glu Phe Asp Phe Leu Ile Lys Gly Gln 50 55 60 Phe Leu Arg Met ProLeu Asp Lys His Met Glu Met Glu Asn Val 65 70 75 Ser Ser Glu Glu Val ValGlu Ile Glu Tyr Val Glu Lys Tyr Thr 80 85 90 Ala Pro Gln Pro Glu Gln CysMet Phe His Asp Asp Trp Ile Ser 95 100 105 Ser Ile Lys Gly Ala Glu GluTrp Ile Leu Thr Gly Ser Tyr Asp 110 115 120 Lys Thr Ser Arg Ile Trp SerLeu Glu Gly Lys Ser Ile Met Thr 125 130 135 Ile Val Gly His Thr Asp ValVal Lys Asp Val Ala Trp Val Lys 140 145 150 Lys Asp Ser Leu Ser Cys LeuLeu Leu Ser Ala Ser Met Asp Gln 155 160 165 Thr Ile Leu Leu Trp Glu TrpAsn Val Glu Arg Asn Lys Val Lys 170 175 180 Ala Leu His Cys Cys Arg GlyHis Ala Gly Ser Val Asp Ser Ile 185 190 195 Ala Val Asp Gly Ser Gly ThrLys Phe Cys Ser Gly Ser Trp Asp 200 205 210 Lys Met Leu Lys Ile Trp SerThr Val Pro Thr Asp Glu Glu Asp 215 220 225 Glu Met Glu Glu Ser Thr AsnArg Pro Arg Lys Lys Gln Lys Thr 230 235 240 Glu Gln Leu Gly Leu Thr ArgThr Pro Ile Val Thr Leu Ser Gly 245 250 255 His Met Glu Ala Val Ser SerVal Leu Trp Ser Asp Ala Glu Glu 260 265 270 Ile Cys Ser Ala Ser Trp AspHis Thr Ile Arg Val Trp Asp Val 275 280 285 Glu Ser Gly Ser Leu Lys SerThr Leu Thr Gly Asn Lys Val Phe 290 295 300 Asn Cys Ile Ser Tyr Ser ProLeu Cys Lys Arg Leu Ala Ser Gly 305 310 315 Ser Thr Asp Arg His Ile ArgLeu Trp Asp Pro Arg Thr Lys Asp 320 325 330 Gly Ser Leu Val Ser Leu SerLeu Thr Ser His Thr Gly Trp Val 335 340 345 Thr Ser Val Lys Trp Ser ProThr His Glu Gln Gln Leu Ile Ser 350 355 360 Gly Ser Leu Asp Asn Ile ValLys Leu Trp Asp Thr Arg Ser Cys 365 370 375 Lys Ala Pro Leu Tyr Asp LeuAla Ala His Glu Asp Lys Val Leu 380 385 390 Ser Val Asp Trp Thr Asp ThrGly Leu Leu Leu Ser Gly Gly Ala 395 400 405 Asp Asn Lys Leu Tyr Ser TyrArg Tyr Ser Pro Thr Thr Ser His 410 415 420 Val Gly Ala 2 2098 DNA Homosapiens misc_feature Incyte ID No 1312429 2 tagaagggca acctcgtgctttctgcagag gagaccggag ggcagaaggc agagtccagg 60 cttagactgc agttcctcgcttacctgtgc agtctaattt tgagctgcct ctttgtagtc 120 ttaaaaggca ggagcttcgtgttgtgggtc tgctaacccg tacgtttccg tgggcaagtc 180 gtgtgtactc ctcgccatggctcagctcca aacacgcttc tacactgata acaagaaata 240 tgccgtagat gatgttcccttctcaatccc tgctgcctct gaaattgccg accttagtaa 300 catcatcaat aaactactaaaggacaaaaa tgagttccac aaacatgtgg agtttgattt 360 ccttattaag ggccagtttctgcgaatgcc cttggacaaa cacatggaaa tggagaacgt 420 ctcatcagaa gaagttgtggaaatagaata cgtggagaag tatactgcac cccagccaga 480 gcaatgcatg ttccatgatgactggatcag ttcaattaaa ggggcagagg aatggatctt 540 gactggttct tatgataagacttctcggat ctggtccttg gaaggaaagt caataatgac 600 aattgtggga catacggatgttgtaaaaga tgtggcctgg gtgaaaaaag atagtttgtc 660 ctgcttatta ttgagtgcttctatggatca gactattctc ttatgggagt ggaatgtaga 720 gagaaacaaa gtgaaagccctacactgctg tagaggtcat gctggaagtg tagattctat 780 agctgttgat ggctcaggaactaaattttg cagtggctcc tgggataaga tgctaaagat 840 ctggtctaca gtccctacagatgaagaaga tgaaatggag gagtccacaa atcgaccaag 900 aaagaaacag aagacagaacagttgggact aacaaggact cccatagtga ccctctctgg 960 ccacatggag gcagtttcctcagttctgtg gtcagatgct gaagaaatct gcagtgcatc 1020 ttgggaccat acaattagagtgtgggatgt tgagtctggc agtcttaagt caactttgac 1080 aggaaataaa gtgtttaattgtatttccta ttctccactt tgtaaacgtt tagcatctgg 1140 aagcacagat aggcatatcagactgtggga tccccgaact aaagatggtt ctttggtgtc 1200 gctgtcccta acgtcacatactggttgggt gacatcagta aaatggtctc ctacccatga 1260 acagcagctg atttcaggatctttagataa cattgttaag ctgtgggata caagaagttg 1320 taaggctcct ctctatgatctggctgctca tgaagacaaa gttctgagtg tagactggac 1380 agacacaggg ctacttctgagtggaggagc agacaataaa ttgtattcct acagatattc 1440 acctaccact tcccatgttggggcatgaaa gtgaacaata atttgactat agagattatt 1500 tctgtaaatg aaattggtagagaaccatga aattacatag atgcagatgc agaaagcagc 1560 cttttgaagt ttatataatgttttcaccct tcataacagc taacgtatca ctttttctta 1620 ttttgtattt ataataagataggttgtgtt tataaaatac aaactgtggc atacattctc 1680 tatacaaact tgaaattaaactgagtttta catttctctt taaaaaaaaa aaaagattaa 1740 ggaaaaaagg tgatatggaaacctagtggg gaatttaacg gtaaatagaa ggggggccgt 1800 tctggggggt ccatcttttaggtacgcggg attgcggggt cctagctctc tctagggggt 1860 ccccaatttc agatcaggggggggtggttt taaaggtgcg gggcttgggg aaaccctggg 1920 ggtttcccaa ttttaagggcctttgggggg aatttcccct ttttggcaca atgggggggt 1980 tattgccgga agggcgcccccaccggttgg ccccgtctcc aaaaatttgg ggggcccctg 2040 aaggggggag ttggggcggcgcccctttta gggggcggtt tatatggccg gggggggg 2098 3 460 PRT Saccharomycescerevisiae misc_feature GenBank ID No g1928989 3 Met Thr Glu Asp Lys SerGln Val Lys Ile Arg Phe Phe Thr Arg 1 5 10 15 Glu Lys Asp Glu Leu LeuHis Val Gln Asp Thr Pro Met Tyr Ala 20 25 30 Pro Ile Ser Leu Lys Arg TyrGly Leu Ser Glu Ile Val Asn His 35 40 45 Leu Leu Gly Ser Glu Lys Pro ValPro Phe Asp Phe Leu Ile Glu 50 55 60 Gly Glu Leu Leu Arg Thr Ser Leu HisAsp Tyr Leu Thr Lys Lys 65 70 75 Gly Leu Ser Ser Glu Ala Ser Leu Asn ValGlu Tyr Thr Arg Ala 80 85 90 Ile Leu Pro Pro Ser Tyr Leu Asn Ser Phe SerAsn Glu Asp Trp 95 100 105 Val Ser Ser Leu Asp Val Gly Asp Gly Ser LysHis Ile Ile Ser 110 115 120 Gly Ser Tyr Asp Gly Ile Val Arg Thr Trp AspLeu Ser Gly Asn 125 130 135 Val Gln Lys Gln Tyr Ser Gly His Ser Gly ProIle Arg Ala Val 140 145 150 Lys Tyr Ile Ser Asn Thr Arg Leu Val Ser AlaGly Asn Asp Arg 155 160 165 Thr Leu Arg Leu Trp Lys Thr Lys Asn Asp AspLeu Lys Leu Thr 170 175 180 Ser Gln Gln Gln Ala Gln Glu Asp Asp Asp AspGlu Val Asn Ile 185 190 195 Glu Asp Gly Lys Thr Leu Ala Ile Leu Glu GlyHis Lys Ala Pro 200 205 210 Val Val Ser Ile Asp Val Ser Asp Asn Ser ArgIle Leu Ser Ala 215 220 225 Ser Tyr Asp Asn Ser Ile Gly Phe Trp Ser ThrIle Tyr Lys Glu 230 235 240 Met Thr Val Val Asp Pro Leu Glu Asp Ile AsnAsn Pro Asn Asn 245 250 255 Lys Ile Ser Thr Ala Ala Arg Lys Arg Arg LysLeu Thr Met Lys 260 265 270 Asp Gly Thr Ile Arg Arg Arg Ala Pro Leu SerLeu Leu Glu Ser 275 280 285 His Thr Ala Pro Val Glu Gln Val Ile Phe AspSer Thr Asp Asn 290 295 300 Thr Val Gly Tyr Ser Val Ser Gln Asp His ThrIle Lys Thr Trp 305 310 315 Asp Leu Val Thr Ala Arg Cys Ile Asp Thr ArgThr Thr Ser Tyr 320 325 330 Ser Leu Leu Ser Ile Ala Gln Leu Ser Thr LeuAsn Leu Leu Ala 335 340 345 Cys Gly Ser Ser Ala Arg His Ile Thr Leu HisAsp Pro Arg Val 350 355 360 Gly Ala Ser Ser Lys Val Thr Gln Gln Gln LeuIle Gly His Lys 365 370 375 Asn Phe Val Ser Ser Leu Asp Thr Cys Pro GluAsn Glu Tyr Ile 380 385 390 Leu Cys Ser Gly Ser His Asp Gly Thr Val LysVal Trp Asp Val 395 400 405 Arg Ser Thr Ser Pro Met Tyr Thr Ile Thr ArgGlu Asp Lys Ser 410 415 420 Val Gln Lys Gly Val Asn Asp Lys Val Phe AlaVal Lys Trp Ala 425 430 435 Glu Lys Val Gly Ile Ile Ser Ala Gly Gln AspLys Lys Ile Gln 440 445 450 Ile Asn Lys Gly Asp Asn Ile Phe Lys Asn 455460

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO:1, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical to the amino acid sequence of SEQID NO:1, c) a biologically active fragment of a polypeptide having theamino acid sequence of SEQ ID NO:1, and d) an immunogenic fragment of apolypeptide having the amino acid sequence of SEQ ID NO:1.
 2. Anisolated polypeptide of claim 1 comprising the amino acid sequence ofSEQ ID NO:1.
 3. An isolated polynucleotide encoding the polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding the polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 comprising thepolynucleotide sequence of SEQ ID NO:2.
 6. A recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotide ofclaim
 3. 7. A cell transformed with a recombinant polynucleotide ofclaim
 6. 8. A transgenic organism comprising a recombinantpolynucleotide of claim
 6. 9. A method of producing a polypeptide ofclaim 1, the method comprising: a) culturing a cell under conditionssuitable for expression of the polypeptide, wherein said cell istransformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:1.
 11. Anisolated antibody which specifically binds to a polypeptide of claim 1.12. An isolated polynucleotide selected from the group consisting of: a)a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2,b) a polynucleotide comprising a naturally occurring polynucleotidesequence at least 90% identical to the polynucleotide sequence of SEQ IDNO:2, c) a polynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional HMAP,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HMAP, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HMAP, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, the methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Amethod for a diagnostic test for a condition or disease associated withthe expression of HMAP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HMAP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, further comprising a label.
 35. A method ofdiagnosing a condition or disease associated with the expression of HMAPin a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:1, or an immunogenicfragment thereof, under conditions to elicit an antibody response, b)isolating antibodies from the animal, and c) screening the isolatedantibodies with the polypeptide, thereby identifying a polyclonalantibody which specifically binds to a polypeptide comprising the aminoacid sequence of SEQ ID NO:1.
 37. A polyclonal antibody produced by amethod of claim
 36. 38. A composition comprising the polyclonal antibodyof claim 37 and a suitable carrier.
 39. A method of making a monoclonalantibody with the specificity of the antibody of claim 11, the methodcomprising: a) immunizing an animal with a polypeptide consisting of theamino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof,under conditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which specifically binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO:1.
 40. A monoclonalantibody produced by a method of claim
 39. 41. A composition comprisingthe monoclonal antibody of claim 40 and a suitable carrier.
 42. Theantibody of claim 11, wherein the antibody is produced by screening aFab expression library.
 43. The antibody of claim 11, wherein theantibody is produced by screening a recombinant immunoglobulin library.44. A method of detecting a polypeptide comprising the amino acidsequence of SEQ ID NO:1 in a sample, the method comprising: a)incubating the antibody of claim 11 with the sample under conditions toallow specific binding of the antibody and the polypeptide, and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide comprising the amino acid sequence of SEQ IDNO:1 in the sample.
 45. A method of purifying a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with the sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising the amino acid sequence ofSEQ ID NO:1.
 46. A microarray wherein at least one element of themicroarray is a polynucleotide of claim
 13. 47. A method of generatingan expression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.